Enhanced glycolysis and suppression of mitochondrial metabolism are features of the Warburg phenomenon in hepatocarcinoma and other cancers. Metabolites enter and exit mitochondria through one channel in the outer membrane: the voltage dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation and decreases glycolysis. Based on exciting new preliminary data, we further hypothesize that VDAC-tubulin antagonists, including erastin, reverse tubulin- dependent VDAC inhibition with consequent mitochondrial hyperpolarization, increased ROS generation and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antagonists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, phospho-AMP kinase, and rates of respiration and glycolysis) in human hepatocarcinoma cells (HepG2, Huh7 and FOCUS). We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane potential (??) and the glycolytic phenotype. Erastin-like compounds that emerged from high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, further evaluated for effects on cellular bioenergetics and used to create a pharmacophore. In Specific Aim 2, we will determine if protein kinase A (PKA) agonists and PKA inhibitors alter the bioenergetics of Huh7, FOCUS and HepG2 cells in the presence and absence of erastin/VDAC- tubulin antagonists and in VDAC isoform double knockdown cells. We will also assess the effects of PKA overexpression/silencing on cellular bioenergetics. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation. In Specific Aim 3 we will determine mechanisms of erastin-induced ROS formation that leads to cell death. We expect that erastin and other VDAC-tubulin antagonists will increase mitochondrial metabolism, ?? and ROS formation, leading to the mitochondrial permeability transition and mitochondrial bioenergetic failure, culminating in cell death. Ultimately, we expect that antagonizing VDAC- tubulin interaction will suppress tumor growth in vivo. Overall, the project will generate fundamental new knowledge on mechanisms underlying suppression of mitochondrial metabolism in hepatocarcinoma cells and identify new agents that block VDAC-tubulin interaction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotoxic oxidative stress.